Brief Freezing Steps Lead to Robust Immunofluorescence in the Drosophila Nervous System

Abstract

Drosophila melanogaster possesses a complex nervous system, regulating sophisticated behavioral outputs, that serves as a powerful model for dissecting molecular mechanisms underlying neuronal function and neurodegenerative disease. Immunofluorescence techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling and, eventually, degradation. However, basic immunostaining techniques do not always result in efficient antibody penetration through the brain, and supplemental techniques to enhance permeability can compromise structural integrity, altering spatial organization. Here, slow freezing of brains is shown to facilitate antibody permeability without loss of antibody specificity or brain integrity. To demonstrate the advantages of this freezing technique, the results of two commonly used permeation methods – detergent-based and partial proteolytic digestion – are compared.

LIM-kinase 1 effects on memory abilities and male courtship song in Drosophila depend on the neuronal type

“…5-6-day-old imago males were anesthetized by freezing. The brains were prepared in PBS buffer (pH 7.5) using needlesharp tweezers (Merck, T4412), fixed in 4 % paraformaldehyde in PBS for 1 h at RT and stained according to (Thapa et al, 2019), without a freezing stage. Antibodies were diluted in PBT (0.2 % Tween 20, 5 % BSA in PBS) as 1:200, for anti-CSP -1:20.…”

cd1 Mutation in Drosophila Affects Phenoxazinone Synthase Catalytic Site and Impairs Long-Term Memory

“…The brains of 5-day-old and 29-day-old adult Drosophila males were isolated in PBS buffer (pH 7.5) using fine needle-sharp style #5 tweezers (Merck, T4412) and fixed in 4% PFA on PBS for 1 h at RT, according to [ 70 ], without the freezing step before staining. The following antibodies were used: primary: rabbit anti- Drosophila tyrosine hydroxylase (TH) antibody (Abcam, ab128249; dilution 1:200), mouse anti- Drosophila cysteine string protein (CSP) antibody (courtesy of E. Buchner, Germany; dilution 1:20); secondary: goat anti-rabbit Alexa Fluor 633 (Invitrogen, A21071; 1:200), goat anti-mouse Alexa Fluor 488 (Invitrogen, A32723; 1:200).…”